Our long-range goal is to understand how chromosome structure affects DNA replication and gene expression. The present proposal focuses on how DNA topoisomerases and supercoiling affect gene expression. We have shown that two topoisomerases are involved in determining the level of DNA supercoiling in bacteria and we now propose to address three questions: 1) can special features of DNA secondary structure be identified near genes whose expression is affected by superhelical density? 2) does DNA gyrase bind to DNA more frequently near actively transcribing genes, and if so, where are the binding sites? and 3) is the expression of a large number of genes affected by changes in supercoiling? Our general strategy involves perturbing the normal level of supercoiling inside cells using topoisomerase mutations, cloned topoisomerase genes and inhibitors of gyrase. DNA secondary structures will be detected by their sensitivity to cleavage by single strand-specific nucleases, gyrase locations will be identified by oxolinic acid-induced DNA cleavage and expression of specific genes by radioactive labeling of proteins that are then identified by gel electrophoresis. Understanding the function of DNA supercoiling is one of the central issues in bacterial chromosome biology and we expect the information gained from this study to form part of the knowledge base necessary to understand and hopefully cure, genetic diseases.